Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.
Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.
Takakusagi
Yoichi
Y
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Kuramochi
Kouji
K
Takagi
Manami
M
Kusayanagi
Tomoe
T
Manita
Daisuke
D
Ozawa
Hiroko
H
Iwakiri
Kanako
K
Takakusagi
Kaori
K
Miyano
Yuka
Y
Nakazaki
Atsuo
A
Kobayashi
Susumu
S
Sugawara
Fumio
F
Sakaguchi
Kengo
K
D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics* / Q000378:metabolism; D015374:Biosensing Techniques / Q000295:instrumentation; D002166:Camptothecin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D000077146:Irinotecan; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000378:metabolism
D000972:Antineoplastic Agents, Phytogenic; D002352:Carrier Proteins; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D006046:Gold; D000077146:Irinotecan; D022021:Tacrolimus Binding Proteins; D002166:Camptothecin